“Transforming growth factor-beta” (TGF-β) represents a family of proteins, which are evolutionarily highly conserved, and affect a broad spectrum of cell types. TGF-β was first identified as a factor, which permitted anchorage-independent growth of primary cell cultures. In vivo, TGF-β promotes the deposition of connective tissue, and induces cell growth of mesenchymal origin. TGF-β affects the proliferation and differentiation of the cells of immune system including hematopoietic stem cells (see Ohta et al., Nature (1987), 329:539) and NK cells (see Rook et al., J. Immunol. (1986) 136:3916). TGF-β may also be administered to suppress hyperproliferation, such as cancer and leukemia (see U.S. Pat. No. 4,816,442).
“Bone morphogenetic protein (BMP)” was extracted from demineralized bone using urea or guanidine hydrochloride and reprecipitated according to the disclosures in the U.S. Pat. Nos. 4,294,753 and 4,455,256. Seyedin and Thomas reported in U.S. Pat. No. 4,434,094 the partial purification of a bone generation-stimulating, bone-derived protein by extraction with chaotropic agents, and recovery of the activity from a fraction adsorbed to CMC at pH 4.8. The new protein fraction was termed “osteogenic factor” and was characterized as having a molecular weight below about 30,000 daltons.
Activins are dimeric proteins, representing a family of proteins structurally related to TGF-β1, and similar to inhibins. Inhibins are heterodimers of the activin subunits and a separate activin subunit. Activins have been shown to stimulate the release of follicle stimulating hormone (see W. Vale et al., Nature (1986), 321:776-79), insulin secretion from pancreatic islets (see Y. Totsuka et al., Biochem. & Biophys. Res. Comm. (1988) 156:335-39), and erythroid and multipotential progenitor cell colony formation in bone marrow culture (see J. Yu et al., Nature (1987) 330:765-67) and induce formation of endochondral bone in vivo (see M. E. Joyce et al., J. Cell Biol. (1990) 110:2195-2207).
Bone sialoprotein (BSP) is a highly glycosylated and sulphated phosphoprotein that is found exclusively in mineralized connective tissues. Polyglutamic acid motifs with the ability to bind hydroxyapatite and cell-surface integrins. BSP has the biophysical and chemical functionalities of a bone nucleator. The hydroxyapatite-binding polyglutamic acid sequence provide bifunctional entites through which BSP may mediate the targeting and attachment of normal and metastasizing cells to the bone surface (see Ganes et al., Bone Sialoprotein, Critical Reviews in Oral Biology and Medicine, 10(1):79-98).
There are several references in the art related to proteins modified by covalent conjugation to polymers, to alter the solubility, antigenicity and biological clearance of the protein (see U.S. Pat. Nos. 4,261,973, 4,301,144, 4,179,337, and 4,830,847). CA2102808, published on 11 Nov. 1992, disclosed a composition comprising a bone growth factor and a targeting molecule having affinity for a tissue of interest, where the bone growth factor and targeting molecule are chemically conjugated to a cross-linker. The cross-linker is preferably a synthetic hydrophilic polymer. The molecules preferably have an affinity for bone. The bone growth factor is preferebly TGF-β, Activin, bone morphogenic protein (BMP), or bone sialoprotein (BSP). The tissue of interest includes bone, cartilage, or other tissues or cell types to which bone growth factors may be targeted. The compositions are intended for use in augmentation of bone formation and repairing or treating bone loss, which are normally found in osteoporosis, osteoarthritis, or age-related loss of bone mass.
It was known that the absorption of calcium in human body basically followed two paths: the active transport and the passive transport. The active transport path is mainly controlled by the regulation of vitamin D and various hormones, in which calcium is absorbed in the upper small intestine against a concentration gradient, while in the passive transport path, calcium in the lower small intestine is absorbed following a concentration gradient. The ratio of the passive transport to the lower small intestine is overwhelmingly high when the soluble calcium is available in abundant. The active transport path cannot rise above a certain amount even if the concentration of soluble calcium increases, while the passive transport rises as the concentration of soluble calcium in the intestine increases. The rate of calcium absorption in the intestines has been reported to be in the range of 10-50%. Casein phosphopeptide (CPP), an enzymatic degradation product of the milk protein casein in gut, accelerates the absorption of calcium (see U.S. Pat. No. 5,447,732 published on 5 Sep. 1995) by raising the concentration of soluble calcium in the small intestine. Calcium is maintained in a soluble state by the complex coordination of the phosphate groups of phosphoserine and carboxylate groups of the acidic amino acids contained in CPP.